CN111826364A

CN111826364A - Disease and insect pest resistance related gene and application thereof

Info

Publication number: CN111826364A
Application number: CN201910242246.2A
Authority: CN
Inventors: 苗雪霞; 王美玲; 时振英
Original assignee: Center for Excellence in Molecular Plant Sciences of CAS
Current assignee: Center for Excellence in Molecular Plant Sciences of CAS
Priority date: 2019-03-28
Filing date: 2019-03-28
Publication date: 2020-10-27
Anticipated expiration: 2039-03-28
Also published as: CN111826364B

Abstract

The invention relates to a disease and insect pest resistance related gene and application thereof. The invention discloses a novel gene from gramineae, which is named as OsRLCK239.1 and has the functions of adjusting plant type characters of plants and improving the capability of the plants to resist plant diseases and insect pests. The invention can be applied to the variety improvement of plants.

Description

Disease and insect pest resistance related gene and application thereof

Technical Field

The invention belongs to the fields of molecular biology and botany, and particularly relates to a disease and pest resistance related gene and application thereof.

Background

The diseases and insect pests of grain crops are the most serious factors threatening the safety of grain production, and the significance of cultivating the crop varieties with disease and insect pests resistance is great.

Intracytoplasmic receptor kinases (RLCK) are receptor-like kinases that lack an extracellular binding ligand domain, most of which contain only a Ser/Thr kinase domain, while other intracytoplasmic receptor kinases also contain an LRR, EGF, WD40 or transmembrane domain. There are 149 and 379 intracytoplasmic receptor kinases in arabidopsis and rice, respectively; based on sequence homology, arabidopsis and rice intracytoplasmic receptor kinases are divided into 17 subgroups. Intracytoplasmic receptor kinases have become a major class of signaling proteins that can respond to biotic or abiotic stress and are involved in many elements of plant growth and development. By associating with immunoreceptor-like Kinases (RLKs), intracytoplasmic Receptor Kinases regulate multiple downstream signaling nodes to coordinate the complex defense response of plants against microbial pathogens. Members within subfamily VII and XII of the many members of the intracytoplasmic receptor kinases have been studied relatively much, primarily in the PTI, ETI and Brassinolide (BR) signaling pathways, and no such enzymes have been studied in rice.

In rice as a food crop, 120 intracytoplasmic receptor kinases are predicted to participate in biotic and abiotic stress, and at present, members of the VIIa subgroup of intracytoplasmic receptor kinases participate in the defense of plant diseases. OsRLCK57, OsRLCK107, OsRLCK118, OsRLCK176 and OsRLCK102 in rice intracytoplasmic receptor kinase VIIa subfamily can negatively regulate BR signals and positively regulate XA 21-mediated resistance to Xanthomonas campestris (Xoo), and disease resistance mechanisms are mainly PTI immune responses induced by positive regulation of chitin and Peptidoglycan (PGN), including active oxygen production and defense gene expression. Plants that down-regulate OsRLCK55 or OsRLCK185 are more sensitive to Xoo; OsRLCK278(BROAD-SPECTRUM RESISTANCE 1, BSR1) belongs to RLCK-VIIb members, can positively regulate the RESISTANCE of bacterial blight and rice blast, and is recently proved to be a kinase with the capacity of tyrosine and serine/threonine double phosphorylation. There is no report in rice that RLCK participates in the ETI immune pathway.

The diseases and insect pests of gramineous plants are the most serious factors threatening the safety of grain production, and the significance of cultivating plant varieties with disease and insect pests resistance is great. The field environment is complex, the plants are simultaneously affected by various diseases and pests, and the optimal characters of the plant varieties are stress resistance and yield guarantee, so the agronomic characters and the stress resistance characters are considered in the research. The art is in need of research useful for improving agronomic traits and stress resistance traits of plants.

Disclosure of Invention

The invention aims to provide a disease and insect resistance related gene and application thereof.

In a first aspect of the invention, there is provided a use of osrlck239.1 or a homologue or a modulator thereof for: improving the ability of plants to resist diseases and insect pests or preparing plants with improved ability to resist diseases and insect pests; or improving the plant type character of the plant or preparing the plant with improved plant type.

In a preferred embodiment, the pests include: bacteria; preferably, the bacteria comprise: bacterial blight (Xanthomonas oryzae pv. oryzae, Xoo), bacterial leaf spot (Xanthomonas oryzae pv. Oryzicola, oc), bacterial brown spot (Pseudomonas syringae pv. syringae VanHoll) and bacterial basal rot (Erwinia chrysanthemi pv. zeae (Sabet) Victria) of rice, etc.

In another preferred embodiment, the pests include: an insect; preferably, the insects are hemipteran insects; more preferably insects of the family Nilaparvata, such as Nilaparvata lugens, Sogatella furcifera and Laileria laevigata; more preferably, OsRLCK239.1 or a homologue thereof improves the ability of a plant to resist a pest by reducing the feed intake of an insect.

In another preferred embodiment, the plant type trait of said modified plant or the production of a plant type modified plant comprises: reducing the plant height of the plant or preparing the plant with reduced plant height; promoting the leaf erection of plants or preparing plants with upright leaves; or increasing the aspect ratio of the plant grain or making a plant with increased grain aspect ratio.

In another preferred example, the OsRLCK239.1 is: (a) 2, as shown in SEQ ID NO; or (b) a protein derived from (a) and having the same function as the protein of (a) and formed by substituting, deleting or adding one or more (e.g., 1 to 20; preferably 1 to 10; more preferably 1 to 5) amino acid residues in the amino acid sequence shown in SEQ ID NO. 2; or (c) a protein derived from (a) having an amino acid sequence which is more than 80% (preferably more than 85%, more preferably more than 90%, more preferably more than 95%, more preferably 98% or 99%) identical to the amino acid sequence defined in (a) and which is functionally identical to the protein of (a); or (d) a protein fragment of SEQ ID NO 2 having the function of the protein of (a).

In another preferred embodiment, the plant comprises: a gramineous plant; preferably, the gramineae comprises: rice, sorghum, corn, barley, wheat, oats, rye.

In another preferred example, the up-regulator of OsRLCK239.1 comprises: an expression construct, expression cassette or expression vector of OsRLCK239.1; a molecule that interacts with OsRLCK239.1 to promote its expression or activity.

In another aspect of the present invention, there is provided a method for increasing the ability of a plant to resist a pest or improving the plant type of a plant, or for producing a plant having increased ability to resist a pest or improved plant type, the method comprising: increasing expression or activity of OsRLCK239.1 or a homologue thereof in a plant.

In a preferred embodiment, the method comprises the following steps: transferring a polynucleotide or construct (e.g., an expression vector) encoding OsRLCK239.1 or a homolog thereof into a plant; or administering an up-regulator of OsRLCK239.1 to the plant, thereby increasing expression or activity of OsRLCK239.1 or a homologue thereof in the plant.

In another preferred embodiment, the method comprises the steps of: (i) providing agrobacterium carrying an expression vector, wherein the expression vector comprises a polynucleotide encoding OsRLCK239.1 or a homolog thereof; (ii) transferring said polynucleotide encoding OsRLCK239.1 or a homologue thereof into a plant using Agrobacterium.

In another preferred embodiment, the plant comprises: a gramineous plant; preferably, the gramineae comprises: rice, wheat, barley, corn, sorghum.

In another aspect of the invention, there is provided a use of OsRLCK239.1 or a homologue thereof or a polynucleotide encoding the same as a molecular marker for identifying a plant type trait or a pest resistance capability of a plant.

In another aspect of the invention, there is provided a plant cell expressing an exogenous osrlck239.1 or a homologue thereof, or an expression cassette comprising an exogenous osrlck239.1 or homologue thereof; preferably, the expression cassette comprises: a promoter, a gene encoding OsRLCK239.1 or a homologue thereof, a terminator; preferably, the expression cassette is comprised in a construct or expression vector.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1, expression profile and kinase properties of OsRLCK239.1. a OsRLCK239.1 expression profile, n is 3; evolutionary analysis results of the bOsRLCK239.1 homologous protein are carried out by a Neighbor-joining Method (Bootstrap methods 500) by using MEGA6.06 software; c, yeast double-hybrid verification of the interaction relationship of OsRLCK239.1, wherein LT represents a Leu and Trp deficient SD yeast culture medium, and LTHA represents a Leu, Trp, His and Ade deficient SD yeast culture medium; d BiLC verifies the self-interaction relationship of OsRLCK239.1, OsRLCK239.1-nLUC + cLUC-OsRLCK239.1 is a verification group, and other combinations are control groups; and e, analyzing the autophosphorylation site distribution of OsRLCK239.1 by using liquid phase mass spectrometry.

FIG. 2, OsRLCK239.1 transgenic plant development phenotype statistical results. a, identifying OsRLCK239.1OE positive plants, wherein n is 3; OsRLCK239.1OE plant in b, c fieldThe plant height phenotype and the statistical result in the plant maturity period are that n is 10; the result of the statistics of dOsRLCK239.1OE internode is that n is 10; e, f, calculating the result of the ear length picture and the ear length, wherein n is 15; the statistical result of the tillering number of the gOsRLCK239.1OE shows that n is 10; h OsRLCK239.1OE, wherein n is 10; iosrlck239.1oe single ear kernel weight; the number of single-ear seeds of j OsRLCK239.1OE; k OsRLCK239.1OE thousand kernel weight statistics. i. j and k, counting 15 rice ears of different lines, wherein the seed counting number is n_wt＝1451，n_oe1＝1340，n_oe6＝1110，n_oe221375. When calculating the significant difference, the statistical analysis is carried out by using a t test, and the significant difference is based on P<0.05，**P<0.01 differentiation.

FIG. 3, the agronomic characters of OsRLCK239.1cas9 plants. a photographs of the fields at flowering stage OsRLCK239.1OE1 and WT; b, knocking out sites of the OsRLCK239.1cas9 knocking out strain, wherein green shows guide sequences, yellow dotted lines show deletion, red shows extra bases, and 67 and 86 show corresponding base position sequence numbers in the OsRLCK239.1 coding gene; c, field photographs of the dosrlck239.1cas9 knockout line; e, performing field statistical analysis on the plant heights of OsRLCK239.1cas9 and wild rice, wherein n is 10; f, statistically analyzing the internode length of OsRLCK239.1cas9-2(cas2) and a wild rice line, wherein n is 15; g, h, i respectively counting the effective tillering number of the wild type, the control and the OsRLCK239.1cas9 knockout strain, wherein n is 10, the length of the rice ear, n is 15 and the tillering number, cCON represents an empty plasmid transgenic plant, and n is 10. Statistical analysis was performed using t-test when calculating significant differences, which were differentiated by P <0.05, P <0.01, no asterisk indicating no significant difference.

FIG. 4, insect-resistant phenotype and mechanism of OsRLCK239.1OE plants. a, the expression of the OsRLCK239.1 gene after wild-type rice is treated by brown planthopper, and the significance analysis is carried out by using t test (P <0.05, P <0.01, n ═ 3); brown planthopper resistant phenotype of different lines of bOsRLCK239.1OE; c, counting the death rate of rice seedlings in a single-plant brown planthopper resistance experiment, wherein 8 rice plants are selected for each strain in the pest resistance experiment and repeated for three times, and n is 3; d, selective experiments of e brown planthopper on OsRLCK239.1OE strain and wild type strain, wherein each strain is repeated for 8 times, and n is 8; f, obtaining the weight increase experimental result of the brown planthopper eating different rice strains, wherein n is 15; g, taking the feed amount of the brown planthopper on different strains, taking the honeydew amount as an analysis object, and taking n as 15; the survival rate of the h-brown planthopper on different strains is counted, the time span is two weeks, and n is 10. Data showed significant differences using t-test (. P <0.05,. P < 0.01).

Fig. 5, osrlck239.1cas9 plants' brown planthopper-like phenotype. a brown planthopper-like phenotype of OsRLCK239.1cas9 plants; b, survival rate of different rice strains in a single-plant insect-resistant experiment, wherein each strain contains 8 rice strains in each experiment, the insect-resistant experiment is repeated for 3 times, and n is 3; survival rates of brown planthopper on OsRLCK239.1cas9 and wild type plants are two weeks, n is 10, d, e respectively represents honeydew amount of brown planthopper on OsRLCK239.1cas9 and wild type plants and weight gain of brown planthopper, and n is 15. Data showed significant differences using t-test (. P <0.05,. P < 0.01).

FIG. 6, OsRLCK239.1OE phenotype against bacterial leaf blight. Pictures of OsRLCK239.1OE anti-bacterial leaf blight after inoculation of bacterial leaf blight for 14 days, and the scale bar is 2 cm; b, the mathematical statistics of lesion length of OsRLCK239.1OE and WT, wherein n is 40; statistical results of disease resistance levels of cOsRLCK239.1OE and WT; growth curves of d.solani 99A in osrlck239.1oe and WT plants, n-3. t-test showed significant differences (. P <0.05,. P < 0.01).

FIG. 7, OsRLCK239.1cas9 plant expression of bacterial blight. Statistics of phenotype (scale bar is 2cm) and lesion length of a, b OsRLCK239.1cas9 plants inoculated with bacterial blight 6 days later, wherein n is 20; c, d and e respectively represent the phenotype (scale bar is 2cm), the statistics of lesion length and the statistics of resistance grade 14 days after the inoculation of the OsRLCK239.1cas9 plants with the bacterial blight, and n is 40. t-test showed significant differences (. P <0.05,. P < 0.01).

FIG. 8, OsRLCK239.1 participating in the signal path. a, b expression change of OsRLCK239.1 in wild rice after exogenous application of 500uM SA and 400uM JA respectively, RNA extraction and detection are carried out at 0, 1, 2 and 4 hours after hormone treatment by using two-week-old Dazhonghua 11 respectively, and n is 3; c, expression amounts of Hi-LOX and AOS2 genes in the d JA synthetic pathway in OsRLCK239.1OE and WT plants, wherein n is 3; e, expression amount of genes PAL and EDS1 in the f SA synthetic pathway in OsRLCK239.1OE plants, wherein n is 3; g the expression level of NPR1 in the SA signaling pathway in OsRLCK239.1OE and WT plants, wherein n is 3; h, i, expression levels of MEK4, MPK3 and MPK6 in the jMAPK signaling pathway in osrlck239.1oe and WT plants, n-3. t test showed significant differences (. P <0.05,. P < 0.01).

Detailed Description

The inventors of the present invention have conducted extensive studies and have revealed that a novel gene derived from gramineae, named osrlck239.1, has the functions of regulating plant type traits of plants and improving the ability of plants to resist diseases and pests. The invention can be applied to the variety improvement of plants.

As used herein, "exogenous" or "heterologous" refers to the relationship between two or more nucleic acid or protein sequences from different sources. For example, a promoter is foreign to a gene of interest if the combination of the promoter and the sequence of the gene of interest is not normally found in nature. A particular sequence is "foreign" to the cell or organism into which it is inserted.

As used herein, the term "expression cassette" refers to a gene expression system comprising all the necessary elements required for expression of a polypeptide of interest (e.g., osrlck239.1 in the present invention), typically comprising the following elements: a promoter, a gene sequence encoding a polypeptide, a terminator; in addition, a signal peptide coding sequence and the like can be optionally included. These elements are operatively connected.

In the early period of research, the inventor finds a protein with undefined function from RNA-seq data, and the protein is named as OsRLCK239.1; and proves that OsRLCK239.1 can form homodimer to generate autophosphorylation; the capacity of over-expressing plants for resisting brown planthopper and white leaf blight is extremely remarkable, and the knockout strain constructed by using the Crisper/cas9 method presents opposite phenotypes of brown planthopper and white leaf blight; and determining that OsRLCK239.1 is probably involved in MAPK cascade reaction, SA and JA signal pathway to regulate the antibiotic capacity of plants.

As used herein, the "plant (crop)" is a plant comprising osrlck239.1 or a homologue thereof; preferably, the plants include, but are not limited to: gramineae, cruciferae, solanaceae, euphorbiaceae, etc. More preferably, the plant is a gramineous plant. For example, the term "plant" includes, but is not limited to: rice, sorghum, corn, barley, wheat, oats, rye, and the like.

The invention also includes fragments, derivatives and analogs of OsRLCK239.1. As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as osrlck239.1 of the invention. A polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more amino acid residues which are conserved or not (preferably conserved amino acid residues) substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide having an additional amino acid sequence fused to the sequence of the polypeptide (e.g., a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the definitions herein.

Any biologically active fragment of OsRLCK239.1 can be used in the invention. Herein, the biologically active fragment of OsRLCK239.1 is meant to be a polypeptide which still retains all or part of the function of the full length OsRLCK239.1. Typically, the biologically active fragment retains at least 50% of the activity of full-length osrlck239.1. Under more preferred conditions, the active fragment is capable of retaining 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of full-length osrlck239.1.

In the present invention, the term "OsRLCK239.1" refers to a polypeptide having the sequence of SEQ ID NO. 2 with OsRLCK239.1 activity. The term also includes variants of the sequence of SEQ ID NO. 2 that have the same function as OsRLCK239.1. These variants include (but are not limited to): deletion, insertion and/or substitution of several (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10, still more preferably 1 to 8, 1 to 5) amino acids, and addition or deletion of one or several (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10, still more preferably 1 to 8, 1 to 5) amino acids at the C-terminal and/or N-terminal (particularly N-terminal). For example, in the art, substitutions with amino acids of similar or similar properties will not generally alter the function of the protein. Also, for example, addition or deletion of one or several amino acids at the C-terminus and/or N-terminus (particularly N-terminus) does not generally alter the function of the protein. The term also includes active fragments and active derivatives of osrlck239.1.

Any protein having high homology with OsRLCK239.1 (such as 50% or more homology with the sequence shown in SEQ ID NO: 2; preferably 60% or more homology; preferably 70% or more homology; preferably 80% or more homology; more preferably 90% or more homology, such as 95%, 98% or 99% homology) and having the same function as OsRLCK239.1 is also included in the present invention.

In the present invention, the term "OsRLCK239.1 conservative variant polypeptide" refers to a polypeptide formed by replacing at most 20, preferably at most 10, more preferably at most 5, and most preferably at most 3 amino acids with amino acids having similar or similar properties, as compared with the amino acid sequence of SEQ ID NO. 2.

It is to be understood that although the osrlck239.1 of the invention is preferably obtained from rice, other polypeptides that are highly homologous (e.g., have greater than 60%, such as 70%, 75%, 80%, 85%, 90%, 95%, or even 98% sequence identity) to osrlck239.1 in rice obtained from other plants are also within the contemplation of the invention, and such polypeptides are also referred to as "homologs" of osrlck239.1. Methods and means for aligning sequence identity are also well known in the art, for example BLAST.

The invention also relates to a polynucleotide sequence encoding the OsRLCK239.1 or conservative variant polypeptide thereof. The polynucleotide may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The sequence of the coding region encoding the mature polypeptide may be identical to the sequence of the coding region shown in SEQ ID NO. 1 or may be a degenerate variant. As used herein, "degenerate variant" refers in the present invention to nucleic acid sequences which encode a protein having SEQ ID NO. 2, but differ from the sequence of the coding region shown in SEQ ID NO. 1.

The polynucleotide encoding the mature polypeptide of SEQ ID NO. 2 comprises: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.

The term "polynucleotide encoding a polypeptide" may be a polynucleotide comprising a sequence encoding the polypeptide, or may be a polynucleotide further comprising additional coding and/or non-coding sequences.

The present invention also relates to variants of the above polynucleotides which encode polypeptides having the same amino acid sequence as the present invention or fragments, analogs and derivatives of the polypeptides. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the polypeptide encoded thereby.

The invention also relates to a vector containing the polynucleotide and a host cell produced by genetic engineering by using the vector or the OsRLCK239.1 coding sequence.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. The transformed plant may be transformed by methods such as Agrobacterium transformation or particle gun transformation, for example, spray method, leaf disk method, rice immature embryo transformation method, etc.

The invention also provides a method of modifying a plant, the method comprising increasing expression of OsRLCK239.1 in the plant. The improved plant comprises: improving the capability of resisting plant diseases and insect pests of plants and improving the plant type characters of the plants. After the function of the OsRLCK239.1 is known, various methods well known to those skilled in the art can be adopted to increase the expression of the OsRLCK239.1. For example, an expression unit (such as an expression vector or a virus) carrying the OsRLCK239.1 gene can be delivered to a target by a way known by the person skilled in the art, and the expression unit can express the OsRLCK239.1 with the activity.

Preferably, there is provided a method of producing a transgenic plant comprising:

(1) transferring the exogenous coding polynucleotide of OsRLCK239.1 into a plant tissue, organ or tissue to obtain the plant tissue, organ or seed of the coding polynucleotide transferred into OsRLCK239.1; and

(2) regenerating the plant tissue, organ or seed which is transferred with the exogenous OsRLCK239.1 encoding polynucleotide obtained in the step (1) into a plant.

Other methods for increasing the expression of the OsRLCK239.1 gene or its homologues are known in the art. For example, expression of the OsRLCK239.1 gene or a homologous gene thereof can be enhanced by driving with a strong promoter. Or the expression of the OsRLCK239.1 gene is enhanced by an enhancer (such as a first intron of a rice waxy gene, a first intron of an Actin gene and the like). Strong promoters suitable for use in the methods of the invention include, but are not limited to: 35s promoter, Ubi promoter of rice and corn, etc.

The methods may be carried out using any suitable conventional means, including reagents, temperature, pressure conditions, and the like.

Based on the new discovery of the inventor, the invention also provides the application of the up-regulator OsRLCK239.1, which is used for improving the plant disease and insect pest resistance or preparing the plant with improved disease and insect pest resistance; or improving the plant type character of the plant or preparing the plant with improved plant type.

As used herein, the up-regulator of osrlck239.1 includes promoters, agonists, and the like. Any substance that can increase the activity of the osrlck239.1 protein, maintain the stability of the osrlck239.1 protein, promote the expression of the osrlck239.1 protein, promote the secretion of the osrlck239.1 protein, prolong the effective action time of the osrlck239.1 protein, or promote the transcription and translation of the osrlck239.1 protein can be used in the present invention, as an effective substance that can be used for plant improvement.

As a preferred mode of the invention, the up-regulator of the OsRLCK239.1 protein comprises (but is not limited to): an expression vector or expression construct which expresses (preferably overexpresses) OsRLCK239.1 after transfer into a cell. Typically, the expression vector comprises a gene cassette comprising a gene encoding OsRLCK239.1 operably linked to expression control sequences. The term "operably linked" or "operably linked" refers to the condition wherein certain portions of a linear DNA sequence are capable of modulating or controlling the activity of other portions of the same linear DNA sequence. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence.

In addition, the invention also relates to a tracking marker for the progeny of the transgenic plant by using OsRLCK239.1 or the coding gene thereof. The invention also relates to a method for identifying the plant type character and the capability of resisting plant diseases and insect pests by detecting the expression condition of OsRLCK239.1 in the plant by using OsRLCK239.1 or the coding gene thereof as a molecular marker. When a plant to be tested is evaluated, whether the expression or mRNA amount in the plant to be tested is higher than the average value of the plants can be known by measuring the expression or mRNA amount of OsRLCK239.1, if the expression or mRNA amount is obviously higher, the plant has higher capability of resisting plant diseases and insect pests, or the plant presents a semi-short stem type on the plant type, or the leaves of the plant are relatively more upright.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Materials and methods

Rice, brown planthopper and white leaf blight

The rice (Oryza sativa) transgenic background material variety is Zhonghua11 (Zhonghua11) provided by the molecular genetics focus laboratory of Chinese academy plants. The rice pest-susceptible variety TN1(Taichung Native 1), the seeds of which are provided by the Rice research institute of China; plant material was cultivated in a climatic chamber. The rice growth conditions are 12h of illumination, 12h of darkness and 28 +/-2 ℃. The field test material is planted in the Songjiang quinque library farm, and the transgenic rice material is planted according to the regulations of related departments and is specially planted in the enclosing wall of the transgenic rice to be tested.

Nilaparvata lugens BPH (brown planthopper of rice)

The original insect source is collected from the Shanghai pine river five-harvesting farm; the TN1 rice seedlings are raised and propagated indoors (temperature 26 +/-2 ℃, illumination 12h and humidity 70-80%).

Xanthomonas (Xanthomonas oryzae pv. oryzae): PXO 99A. Growth was carried out in PSA medium at 28. + -. 2 ℃ in an incubator.

Plasmids

The plasmids of the present invention are shown in Table 1.

TABLE 1

Construction of an evolutionary tree

BLAST was performed on the OsRLCK239.1 protein sequence in Rice database (Rice Genome Annotation Project, RGAP) and evolutionary analysis was performed on the three homologous sequences with the highest scores and the protein sequences downloaded from the functionally reported RLCK gene.

Overexpression vector construction

The coding region sequence of OsRLCK239.1 is amplified and ligated into SpeI and KpnI sites of pCambia1301: 35s vector by using an enzyme digestion and linking method.

The coding sequence (nucleotide sequence) of OsRLCK239.1 is as follows:

ATGAGGTCATCCAGCGATTGCAAGGTTGTGGCGGCGGCGGCGAGGAAGAAGGAGAAGGAGGCGGCGGCGTGGCCGTGGTCGCTGTGGGGGTTCCTCCTGACCGGCTGCCTCGGCGGCGGCGGCGGCGGAGGGAAGAAGAAGAGCGGGGGGAAGAAGGTGCGTCCCCGCGGCGGCGGCGGCGGCCTGCGGCGGCTGTCGTTCACGGACCTGACGGGGGCGGCGGACCAGGACCTGTCGGTGTCGCTGGTGGGGTCCAACCTCCACGTCTTCACCGTCGCCGAGCTCCGCGACGCCACCCGCGGGTTCGTCTCCGGCAACTTCCTCGGCGAGGGCGGCTTCGGGCCGGTCTACAAGGGCCTCGTCGGCGACGGCGTCAAGCCGGGCCTCCGCCCGCAGGCCATCGCCGTCAAGCTCTGGGATCCCGAGGGCGCCCAGGGCCACAAGGAATGGCTGGCAGAGGTGATCTTCCTTGGCCAGCTTCGGCATCCCAACCTGGTGAAGCTGGTCGGCTACTGCTGCGAGGACGAGAACCGCCTCCTCGTCTACGAGTACATGGAGCATGGCAGCCTCGAGAACCACCTCTTCAAACAGATTCCTGCCGTGCTGCCGTGGTCGACCCGATTAAACATCGCGGTTGGCGCCGCGAAGGGTTTGGCGTTCCTCCACGACGCAGAGAAGCCGGTCATCTACCGTGACTTCAAGGCTTCCAACATCCTGCTCGATTCGGATTACAAGGCGAAGCTGTCGGACTTCGGGCTGGCCAAGGACGGGCCGGAGGGGGACGACACCCACGTGTCGACGCGCGTGATGGGCACCCATGGCTACGCCGCGCCGGAGTACATCATGACCGGCCACCTGACGGCGAAGAGCGACGTGTACAGCTTCGGCGTGGTGCTCCTGGAGATCCTGACGGGGCGGCGCGCCGTCGACAAGACGCGGCCGAACAGGGAGCAGAGCCTCGTGGAGTACGCGCGGCCGTGCCTGCGCGACCCGCTCCGGCTCATCCGGATCATGGACCCGGCGCTGGAGGGGCGCTACTCGCCGGCGGCGGCGAGGGAGGCGGCCGCCGTCGCCTACCGGTGCCTCAGCGGGAGCCCCAAGAACCGCCCCGACATGTCCGCCGTCGTCGACGCGCTCGAGCCGCTGCTCGTCGCCACCGACGACGTCCCCCTCGGCCCCGTCGTGCTGTTCGTCGCGCCGGATCAGGAGGCCGACGCCGCCGCCGCCGCCGACGACGACGAGGACGACAAGGCCCGGCGGCGGCAGCGGCGGACGCGGAAGGACGAGCAGCACCGCCGCCGCAGCCGCCTCCGGACGTCGCCCAAGGGCAGCCCGAGGAAGCCCGCCGTCGCCGCCGCTTGCCGGAACGAGGAGTTCTGGGTGTGGCACGTCCCCGCCGACCACAAGGCGTGA

the amino acid sequence of OsRLCK239.1 is as follows:

MRSSSDCKVVAAAARKKEKEAAAWPWSLWGFLLTGCLGGGGGGGKKKSGGKKVRPRGGGGGLRRLSFTDLTGAADQDLSVSLVGSNLHVFTVAELRDATRGFVSGNFLGEGGFGPVYKGLVGDGVKPGLRPQAIAVKLWDPEGAQGHKEWLAEVIFLGQLRHPNLVKLVGYCCEDENRLLVYEYMEHGSLENHLFKQIPAVLPWSTRLNIAVGAAKGLAFLHDAEKPVIYRDFKASNILLDSDYKAKLSDFGLAKDGPEGDDTHVSTRVMGTHGYAAPEYIMTGHLTAKSDVYSFGVVLLEILTGRRAVDKTRPNREQSLVEYARPCLRDPLRLIRIMDPALEGRYSPAAAREAAAVAYRCLSGSPKNRPDMSAVVDALEPLLVATDDVPLGPVVLFVAPDQEADAAAAADDDEDDKARRRQRRTRKDEQHRRRSRLRTSPKGSPRKPAVAAACRNEEFWVWHVPADHKA*

BiLC vector construction

pCambia1300-nLUC and pCambia1300-cLUC vectors were single-stranded using KpnI and SalI, and the coding sequence for OsRLCK239.1 was ligated into the vector pCambia1300-nLUC and vector pCambia1300-cLUC, respectively, using homologous recombination.

Yeast double hybrid vector construction

The OsRLCK239.1 coding sequence is respectively connected to EcoRI and BamHI sites of pADT7 and pBKT7 by a homologous recombination method, and 20bp at the 5 'end and the 3' end of the EcoRI and BamHI enzyme cutting sites are respectively selected as homologous arms.

Prokaryotic expression vector construction

The full length of the coding sequence of OsRLCK239.1 and the coding sequence (193-1413bp) with the signal peptide removed are ligated into EcoRI and BamHI cleavage sites of pGEX4T1-1 by homologous recombination.

CRISPR-Cas9 technology

Designing a Spacer: finding out available identification sequence corresponding to gene number in text library, comparing with cds and genome sequence, selecting coding region near 5 'end, taking NGG as tail, removing NGG plus 8 front segment sequences at 5' end, adding GGCA at F end, adding AAAC at R end, and primer total 20 bp. Inputting the selected primers into a target off-target prediction website CRISPER-P (http:// cbi. hzau. edu. cn/cgi-bin/CRISPR), performing off-target prediction, inputting sgRNA into a primer structure prediction website OligoEvaluater (http:// www.oligoevaluator.com), and clicking Calcute to obtain gene analysis. Finally, an appropriate sgRNA sequence is determined by comprehensively considering Tm (56-62), GC content (45-60%), secondary structure and test results, and the sequence with high score is not necessarily the optimal sequence. According to the final result, sgRNA primers were determined as follows:

sgRNA-OsRLCK239.1F：GGCAGCGTGGCCGTGGTCGCTGTG

sgRNA-OsRLCK239.1R：AAACCACAGCGACCACGGCCACGC

a spacer double-stranded fragment is synthesized and linked to the BsaI site of the pOS-sgRNA to construct pOS-sgRNA-OsRLCK239.1. Then, the vector was ligated into pOS-Cas9 vector to obtain pOS-Cas 9-OsRLCK239.1. Carrying out tissue culture experiment of the transgenic plants.

Insect resistance test of rice single plant

And respectively carrying out insect resistance identification on the single plants at the seedling stage and the tillering stage. Selecting two-week-old rice seedlings with uniform sizes, planting the single seedlings in a 5x5cm small box, inoculating 2-3-year-old brown planthoppers into each seedling, photographing and counting the death rate when one strain is nearly dead, and repeating the experiment for 3 times by repeating 8 strains of the single strain each time. For the insect-resistant identification at the tillering stage, two days after tillering needs to be cut off, insect-resistant identification is carried out again, and 20 2-3 instar brown planthoppers are inoculated. After the inoculation, the rice state needs to be observed every day, and photographing and rice death rate statistics can be carried out in about 5-8 days.

Selectivity experiment of brown planthopper on rice

Selecting rice seedlings with the size of 1 month, putting the rice seedlings in boxes filled with soil with the size of 9 multiplied by 9cm, transplanting wild type ZH11 at the opposite corners of each transgenic line, planting 8 boxes in each transgenic line, and performing a selectivity test on OsRLCK239.1OE1 and OE 6. 15 female adults were picked up and placed in the middle of the box, and the number of brown planthoppers on each rice plant was recorded at 0, 1, 3, 6, 12, 24, 48 hour time points. The total number of brown planthoppers on the same line of plants in the same box is recorded, and the average value of 8 replicates is calculated for comparison.

Brown planthopper honeydew amount and insect weight gain experiment

Respectively putting 5 brown planthopper nymphs of three instars into the weighted wax bags, weighing again to obtain the initial weight of the brown planthopper, hanging the brown planthopper on a plant, weighing again the total weight of the wax bags after two days, and removing the total weight of the nymphs. The weight gain of the brown planthopper and the amount of honeydew secreted by the brown planthopper after the brown planthopper takes food can be calculated.

Brown planthopper survival rate statistics

One month old rice seedlings are respectively planted in small plastic boxes, 15 nymphs of two years old are inoculated to each rice plant, and after two weeks, the number of the surviving nymphs is counted. Each line was replicated 5 strains.

Inoculation of bacterial blight and production of growth curve

1) Taking PXO99A strain stored in refrigerator at-80 deg.C, scratching on PSA plate for 1 time, and culturing at 28 deg.C for 48-72 h.

2) After the monoclonal bacteria grow out, selecting the monoclonal bacteria until the PSA liquid culture medium is shaken for 1-2 days.

3) 100 μ L of the bacterial solution was aspirated to a PSA (15mg/L cephalexin) plate, uniformly spread on a clean bar, and incubated at 28 ℃ for 48 h. And adjusting the number of the drawing boards according to the concentration of the bacterial liquid and the inoculation requirement. The grown leaf blight germ can be used for rice inoculation.

4) The bacterial blight grown on the PSA medium was scraped off with sterile water using an applicator and diluted to an OD of 1.0.

5) The bacterial liquid is dipped by scissors, the leaf apex of the rice is cut off at the position of about 2cm of the leaf apex obliquely downwards, 2-3 tillers are cut for each plant by selecting 3 tillers, and inverted two leaves and inverted three leaves are mainly selected. Dipping in a primary bacterial liquid to cut three blades in a stretching state.

6) After 12-14 days of inoculation, the length of the lesion of the leaf and the total length of the leaf (for the identification of the resistance grade) are measured. The correspondence between the rank and the lesion size is shown in Table 2.

TABLE 2

Rank of	Size of lesion
		0, High Resistance (HR)	No obvious disease spot at the cut
1, anti (R)	The lesion spots are expanded downwards and have a length of 2-3 cm
		3, Moderate (MS)	1/4 with lesion length less than the length of the inoculating leaf
5, middle feeling (MS)	The length of the lesion is 1/4 larger than that of the inoculated leaf but is less than 1/2
		7, feeling (S)	The length of the lesion is 1/2 larger than that of the inoculated leaf but is less than 3/4
9, high feeling (HS)	3/4 with lesion length greater than the length of the inoculated leaf

Production of growth curves

1) A small amount of 95% industrial alcohol is added into the mortar for combustion, and the mixture is cooled to room temperature (the effect is sterilization).

2) Taking 10cm of rice leaves infected with different days, wherein each sample contains 3 leaves, wiping the surface with 75% ethanol, shearing, placing into a mortar, adding a small amount of sterilized quartz sand and 2ml of sterilized water, grinding in an ultra-clean workbench, continuously adding sterile water to 10ml, oscillating, mixing uniformly, and fully releasing thalli.

3) Taking out a small amount of the uniformly oscillated bacterial liquid, performing gradient dilution (generally performing dilution of 100 times, 1000 times and 10000 times), coating a PSA plate containing 15mg/L cephalexin (cephalexin, BBI company), and counting the number of grown clones after growing for 2-3 days, wherein each material is subjected to three repetitions at each time point.

4) Finally, the Bacterial population [ Log (c.f.u./leaf) ] of each material was calculated.

Yeast double-impurity screening storehouse

1) Plasmid BD-HLH61 was transformed into AH109 yeast strain and its self-activation was verified.

2) Selecting larger positive monoclonal of BD-HLH61, placing in 50ml SD/-Trp culture medium, shaking (250-270 rpm) at 30 deg.C for culturing (16-20hr) to OD600 ═ 0.8, centrifuging to collect thallus (1, 000g, 5min), discarding supernatant, re-suspending with 4-5 ml SD/-Trp liquid culture medium, counting bacterial liquid cell number per ml by blood counting plate>1x10⁸。

3) 10ul of the library suspension (Y178) was taken out from 1ml of the library suspension to prepare diluted gradient solutions (1/100, 1/1, 000, 1/10, 000) which were applied to SD/-Leu solid medium for library capacity detection, mixed with AH109(BD-HLH61) resuspended suspension, placed in a 2-L flask, and 45ml of 2 XYPMDA liquid medium (containing 50. mu.g/ml kanamycin antibiotic) was added thereto, and a vial of the library suspension was washed twice with 1ml of 2 XYPMDA medium and then added to the large flask.

4) Culturing at 30 deg.C under low speed shaking (40-60 rpm) for 20-24hr, and stopping shaking when the presence of 'clover' or 'Mickey' like Mating bacteria is detected by 40X microscope.

5) The cells were collected at 1,000 g for 10min, and the 2-L flasks were washed 2 times with 50ml of 0.5XYPDA (containing 50. mu.g/ml of kanamycin antibiotic) liquid medium to collect the cells, and the supernatant was centrifuged again.

6) Finally, the cells were suspended in 10ml of 0.5 XYPMDA/Kan medium to give a total volume of about 11.5 ml.

7) And (3) determination of hybridization efficiency: 30ul of the hybrid bacterial suspension was diluted at 1/10, 1/100, 1/1, 000, and 1/10, 000 and applied to 100mm plates of SD/-Trp, SD/-Leu, and SD/-Leu/-Trp, respectively, and cultured at 30 ℃ for 3-5 days.

8) The remaining plating broth was applied to four medium plates (50-55 plates) in an amount of 200. mu.l per 150mm plate, and cultured at 30 ℃ for 3-5 days.

9) The number of colonies on the plate was counted and the hybridization efficiency was calculated (2-5%).

10) Colonies on the four-lacking plate were transferred to new four-lacking medium, and false positive colonies were removed.

11) Fragment amplification was performed using yeast colony PCR with primers using universal primers T7 and 3' AD for AD vector, purification PCR, and sequencing with T7 primer.

12) Selecting candidate genes from the sequencing result, extracting corresponding yeast plasmids, cotransforming the yeast plasmids and the bait plasmids by a lithium acetate method again, and determining the interaction relationship.

Luciferase complementation assay (BiLC)

Transferring OsRLCK239.1-nLUC, cLUC-OsRLCK239.1, nLUC and cLUC into Agrobacterium GV3101, identifying positive bacteria, transferring the identified bacteria into liquid culture medium, centrifuging, collecting bacteria, washing, centrifuging, incubating with working solution for 1-2hr, and adding OD with single bacteria solution final concentration₆₀₀About 1.0, the tobacco was injected together, 1/4, which was the same piece of tobacco injected in the control group and the experimental group; three days later, luciferase substrate was injected at 30mg/ml, diluted 200-fold and injected (assist in san Ltd., Cat. No. 40901), tobacco leaves were cut off to ensure consistent injection area, and a cold CCD camera was usedFluorescence signals were collected in the dark for 10min, and the presence of fluorescence expression indicated that protein interaction occurred.

Statistics of agricultural characters of field

Measuring the lengths of different internodes of 10 individual stems, and taking the average value as the internode length; the spike head number of each plant is taken as the effective tillering number, and each strain is counted into 10 plants; randomly measuring the length of 15 ears in different strains of plants for statistical analysis; the seed properties such as the number of seeds in a single spike, weight, setting rate, seed length, width, thousand seed weight and the like are analyzed in batches by using an automatic seed test analyzer with ten thousand depths, and each experiment is repeated for 15 times.

In vitro phosphorylation

Phosphorylation assay reaction System (40. mu.l System)

By ddH₂The content of O is supplemented to 40 ul. The reaction was carried out at 30 ℃ for 45 min.

Adding 15ul 4X protein loading Buffer, and decocting at 95 deg.C for 10 min.

Running protein gel, 100V, 80 min.

And then dyeing with Coomassie brilliant blue, decoloring, cutting a target strip, and preparing a mass spectrum sample.

Example 1 expression profiling and homologous Gene analysis of OsRLCK239.1

The inventor finds an unknown functional gene OsRLCK239.1 from the RNA-seq data of the downregulated HLH61 plant. Through analysis, the protein contains a kinase structural domain and belongs to a member of the VIIa subfamily of the cytoplasmic receptor protein kinase (RLCK) (Liu et al, 2011). After the spatiotemporal expression analysis of OsRLCK239.1, the OsRLCK239.1 is also found to be accumulated in a large amount in leaves of a white leaf blight hazard part and leaf sheaths of a brown planthopper feeding part (figure 1 a). The result of evolutionary analysis of OsRLCK239.1 homologous protein is shown in FIG. 1 b. The phenomenon that kinase tends to form homodimers and is subjected to autophosphorylation has been reported in the literature (Han et al, 2014), and the fact that OsRLCK239.1 can form homodimers by itself is verified through yeast double hybrid (figure 1c) and BiLC experiments (figure 1 d); after protein purification by ligating the OsRLCK239.1 gene into a prokaryotic expression vector with a GST tag, and carrying out in vitro phosphorylation experiments, the autophosphorylation sites of OsRLCK239.1 are identified by using a liquid phase mass spectrometry method, and partial Ser (S) and Thr (T) amino acids can be phosphorylated (Table 1) and a main phosphorylation region (figure 1e) is counted, so long as the autophosphorylation region is concentrated in a kinase domain, and a few autophosphorylation sites also exist at the downstream of a glycine aggregation section (figure 1 d). These characteristics prove that OsRLCK239.1 is a serine/threonine protein kinase which can form a dimer by itself and has the function of autophosphorylation.

Example 2 reduction of plant height in OsRLCK239.1OE plants

After determining that the OsRLCK239.1 protein is a functional kinase, constructing a transgenic plant. The coding sequence of OsRLCK239.1 is connected into pCambia1301::35s overexpression vector, and transgenic rice is cultivated by agrobacterium transfection method.

Selecting 3 over-expression positive strains (OsRLCK239.1OE), abbreviated as OE1, OE6 and OE22 (FIG. 2a) by fluorescence quantitative detection; OsRLCK239.1OE plants have significant changes in development phenotype, wherein the changes in plant height are the most significant, and the plant height is counted by taking a picture of a single plant in a field test (figure 2b) and counting the plant height (figure 2c) and the internode length is counted by taking OE1 as a representative (figures 2b and d), wherein the OE1 leaves are more upright, and the phenomenon is more significant in the flowering phase (figure 3 a).

In addition, statistics on yield-related traits were carried out, and it was found that the tillering number (fig. 2g) and the effective tillering number (fig. 2h) were not different from wild type except that the ear length of osrlck239.1oe plants was shortened (fig. 2e, f), the kernel weight per ear (fig. 2i), the kernel number (fig. 2g) and the thousand kernel weight (fig. 2k) were not significantly different, except that there was a difference in OE6 strain, probably because the expression level of osrlck239.1 in OE6 was the highest, and the gene of osrlck239.1 had a dose effect (fig. 2 a); whereas the aspect ratio of osrlck239.1oe grain was significantly higher than wild type, and the area of grain was smaller than wild type (table 3), indicating that the properties of grain were changed, but the quality of grain was still not changed (fig. 2 k).

TABLE 3 OsRLCK239.1OE grain trait

In table 1, the seed properties were examined using a ten thousand depth automatic seed-testing analyzer, and seeds of 15 ears were selected for each line for statistics.

From the above results, the present inventors considered that the OsRLCK239.1OE plant is a transgenic plant having a semi-dwarf plant type without affecting yield.

The inventor constructs a knockout plant of OsRLCK239.1 by using the technology of Crisper/cas9, a guide sequence used for knockout is positioned at the position 67-86 of the coding sequence of OsRLCK239.1, and two mutant strains (OsRLCK239.1cas9) are obtained and are simply named as cas2, cas7 and cas18 respectively, wherein cas2 lacks 14bp, and cas7 and cas18 both have one more T base after the position of 83 bases (figure 3b), and the two mutations can completely change the protein sequence of OsRLCK239.1.

In development traits, the knockout OsRLCK239.1 does not change in plant height (FIG. 3c, d, e), internode length (FIG. 3f), effective tiller number (FIG. 3g), spike length (FIG. 3h) and tiller number (FIG. 3i), and the function of the knockout OsRLCK239.1 is possibly supplemented by the homologous gene redundant with OsRLCK239.1. The agronomic characters of the OsRLCK239.1cas9 plant are not changed.

Example 3 OsRLCK239.1 can positively modulate resistance of rice to Nilaparvata lugens insect pests

Based on the results of the foregoing example 2, the OsRLCK239.1OE plant can dwarf the rice plant, and the present inventors further analyzed the effect of OsRLCK239.1 on Nilaparvata lugens.

The expression change of OsRLCK239.1 in wild-type rice treated by qRT-PCR detection is firstly used, and the result shows that the OsRLCK239.1 can respond to the feeding activity of brown planthopper, and the expression level is obviously improved at 3, 7 and 12 hours after induction (figure 4 a).

Then, the over-expression OsRLCK239.1 plant is determined to have extremely remarkable capacity of resisting brown planthopper through a single-plant insect-resistant experiment (figure 4b) and a rice mortality statistical result in the experiment (figure 4 c).

In order to clarify the anti-insect mechanism of the gene, a selection experiment of brown planthopper on the rice line is carried out, and the result shows that the OE1 is obviously different from the OE6 at the 6 th hour (figure 4d) of inoculation (figure 4 e).

Honeydew amount assay (fig. 2f), brown planthopper growth status weight gain assay (fig. 2g) and brown planthopper survival assay (fig. 2h) were performed on different lines of osrlck239.1oe and WT, respectively. The results demonstrate that the feed intake of brown planthopper on osrlck239.1oe plants is significantly reduced (fig. 2f), which in turn affects the growth of brown planthopper, but not the survival of brown planthopper. The OsRLCK239.1OE plant achieves an extremely obvious insect-resistant purpose through an antibiotic mechanism which mainly reduces the feeding of brown planthopper, influences the growth of the brown planthopper and is not completely lethal.

In contrast to the insect-resistant phenotype of osrlck239.1oe plants, the osrlck239.1cas9 plants exhibited an insect-susceptible phenotype (fig. 5a), the mortality rate of the osrlck239.1cas9 plants was significantly higher than that of wild-type plants (fig. 5b), the number of brown planthoppers surviving on the osrlck239.1cas9 and wild-type plants (fig. 5c), the honeydew amount (fig. 5d) and the insect weight gain (fig. 5e) were increased in the individual insect-resistant experiments, but there was no significant difference.

The results can prove that OsRLCK239.1 can positively regulate the resistance of rice to brown planthopper.

Example 4 OsRLCK239.1 can positively regulate resistance of rice to bacterial blight disease

In this example, the disease resistance of osrlck239.1 is examined.

A strain 99A with the strongest pathogenicity in Xanthomonas oryzae pv. oryzae, Xoo, one of three diseases of rice is selected to carry out a field disease resistance experiment, after the inoculation for 14 days, the length of each strain is measured by 40 leaves and the length of a disease spot, the disease resistance result is shown in figure 6a-b, and the disease spot length of an OsRLCK239.1OE plant is extremely lower than that of a control plant.

Statistical grading is carried out according to the disease-resistant grade of the bacterial blight in China, the result shows that the resistance of the over-expression OsRLCK239.1 plant to the bacterial blight belongs to a high-resistance grade (figure 6c), and the OE6 strain is close to the immunity grade.

The inventors have made a germ growth curve, which better shows the growth and propagation speed of germs, and the growth capacity of germs in OsRLCK239.1 overexpression plants is significantly lower than that of germs in wild type (FIG. 6 d).

In contrast to the over-expressed plants, the length of the disease spot was measured at 6 days after inoculation, and the OsRLCK239.1cas9 plants were statistically found to exhibit the bacterial blight-susceptible phenotype (FIG. 7a), and the statistical results are shown in FIG. 7 b; however, after 14 days of inoculation, the length of lesion spots of OsRLCK239.1cas9 plants was not significantly different from that of wild type (FIGS. 7 c-e).

The above results indicate that OsRLCK239.1 can positively regulate the resistance of rice to bacterial blight.

Example 5 OsRLCK239.1 negatively regulates SA, JA and MAPK signaling pathways

After confirming the positive regulation effect of OsRLCK239.1 in biological resistance, the inventors further studied the resistance action mechanism of OsRLCK239.1.

Firstly, the hormones SA (salicylic acid) and JA (jasmonic acid) closely related to stress resistance in plants are selected, 500uMSA and 400uM JA are applied externally, after wild-type rice is treated for two weeks, sampling is carried out at time points of 0, 1, 2 and 4h after treatment, after RNA is extracted, the expression level of OsRLCK239.1 is detected by using qRT-PCR, and the OsRLCK239.1 can be obviously increased at the expression level of 1 and 4h after SA treatment (figure 8a), while the expression level of OsRLCK239.1 is obviously inhibited at the expression level of 2h after JA treatment (figure 8 b). The SA and the JA which are applied by external sources have antagonistic effect on the expression of the OsRLCK239.1; the expression levels of the JA synthetic pathway genes Hi-LOX and AOS2 in OsRLCK239.1OE plants were significantly lower than that of the wild type (FIG. 8c, d); the expression level of genes PAL and EDS1 of the SA synthesis pathway and the SA receptor NPR1 in the signaling pathway were also significantly lower than that of the wild type (fig. 8e, f, g); and as RLCK is mostly related to MAPK signaling pathway, the detection shows that the expression level of MEK4 (figure 8h) and MAPK3 (figure 8i) in OsRLCK239.1OE plants is also significantly reduced, while the expression change of MAPK6 is not significant (figure 8 j).

The above results indicate that exogenously applied SA and JA can antagonistically modulate the expression of osrlck239.1, while overexpressing osrlck239.1 can attenuate SA, JA and MAPK signaling pathways. The extremely significant pest-resistant phenotype of OsRLCK239.1OE is shown to be due to the ability to multiple resistance signaling pathways.

Conclusion

The inventor discovers that the new gene OsRLCK239.1 belongs to a receptor intracytoplasmic kinase VIIa subfamily member in the structural characteristics from the original RNA-seq data in a laboratory, can form a dimer by itself and has the capacity of in vitro autophosphorylation. The over-expression OsRLCK239.1 plant can form a semi-short plant type with upright leaves, has no obvious influence on the number of grains per spike and the thousand seed weight, and has extremely obvious capacity of resisting brown planthopper and bacterial blight in addition, wherein the brown planthopper and the bacterial blight are respectively one of the most main pests and diseases of rice. An OsRLCK239.1 knockout plant (OsRLCK239.1cas9) is constructed by using a Crisper/cas9 technology, and the agronomic characters of the OsRLCK239.1cas9 plant are not changed, but the resistance to brown planthopper and white leaf blight is obviously reduced; the expression of OsRLCK239.1 can be induced by exogenously applied SA, the expression of OsRLCK239.1 can be inhibited by exogenously applied JA, and JA, SA and MAPK signal pathways are obviously inhibited in OsRLCK239.1OE plants. The invention discovers that an intracytoplasmic receptor kinase, OsRLCK239.1, can influence the antibiotic performance of rice by influencing SA, JA and MAPK signal paths. The plant of the over-expression OsRLCK239.1 has excellent plant type without influencing yield, has the capability of resisting diseases and insect pests at the same time, and can be used for later molecular breeding to culture a new variety with high resistance and high yield.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Shanghai Life science research institute of Chinese academy of sciences

<120> disease and insect pest resistance related gene and application thereof

<130>190640

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<170>SIPOSequenceListing 1.0

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<213> Rice (Oryza sativa)

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atgaggtcat ccagcgattg caaggttgtg gcggcggcgg cgaggaagaa ggagaaggag 60

gcggcggcgt ggccgtggtc gctgtggggg ttcctcctga ccggctgcct cggcggcggc 120

ggcggcggag ggaagaagaa gagcgggggg aagaaggtgc gtccccgcgg cggcggcggc 180

ggcctgcggc ggctgtcgtt cacggacctg acgggggcgg cggaccagga cctgtcggtg 240

tcgctggtgg ggtccaacct ccacgtcttc accgtcgccg agctccgcga cgccacccgc 300

gggttcgtct ccggcaactt cctcggcgag ggcggcttcg ggccggtcta caagggcctc 360

gtcggcgacg gcgtcaagcc gggcctccgc ccgcaggcca tcgccgtcaa gctctgggat 420

cccgagggcg cccagggcca caaggaatgg ctggcagagg tgatcttcct tggccagctt 480

cggcatccca acctggtgaa gctggtcggc tactgctgcg aggacgagaa ccgcctcctc 540

gtctacgagt acatggagca tggcagcctc gagaaccacc tcttcaaaca gattcctgcc 600

gtgctgccgt ggtcgacccg attaaacatc gcggttggcg ccgcgaaggg tttggcgttc 660

ctccacgacg cagagaagcc ggtcatctac cgtgacttca aggcttccaa catcctgctc 720

gattcggatt acaaggcgaa gctgtcggac ttcgggctgg ccaaggacgg gccggagggg 780

gacgacaccc acgtgtcgac gcgcgtgatg ggcacccatg gctacgccgc gccggagtac 840

atcatgaccg gccacctgac ggcgaagagc gacgtgtaca gcttcggcgt ggtgctcctg 900

gagatcctga cggggcggcg cgccgtcgac aagacgcggc cgaacaggga gcagagcctc 960

gtggagtacg cgcggccgtg cctgcgcgac ccgctccggc tcatccggat catggacccg 1020

gcgctggagg ggcgctactc gccggcggcg gcgagggagg cggccgccgt cgcctaccgg 1080

tgcctcagcg ggagccccaa gaaccgcccc gacatgtccg ccgtcgtcga cgcgctcgag 1140

ccgctgctcg tcgccaccga cgacgtcccc ctcggccccg tcgtgctgtt cgtcgcgccg 1200

gatcaggagg ccgacgccgc cgccgccgcc gacgacgacg aggacgacaa ggcccggcgg 1260

cggcagcggc ggacgcggaa ggacgagcag caccgccgcc gcagccgcct ccggacgtcg 1320

cccaagggca gcccgaggaa gcccgccgtc gccgccgctt gccggaacga ggagttctgg 1380

gtgtggcacg tccccgccga ccacaaggcg tga 1413

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Met Arg Ser Ser Ser Asp Cys Lys Val Val Ala Ala Ala Ala Arg Lys

1 5 10 15

Lys Glu Lys Glu Ala Ala Ala Trp Pro Trp Ser Leu Trp Gly Phe Leu

20 25 30

Leu Thr Gly Cys Leu Gly Gly Gly Gly Gly Gly Gly Lys Lys Lys Ser

35 40 45

Gly Gly Lys Lys Val Arg Pro Arg Gly Gly Gly Gly Gly Leu Arg Arg

50 55 60

Leu Ser Phe Thr Asp Leu Thr Gly Ala Ala Asp Gln Asp Leu Ser Val

65 70 75 80

Ser Leu Val Gly Ser Asn Leu His Val Phe Thr Val Ala Glu Leu Arg

85 90 95

Asp Ala Thr Arg Gly Phe Val Ser Gly Asn Phe Leu Gly Glu Gly Gly

100 105 110

Phe Gly Pro Val Tyr Lys Gly Leu Val Gly Asp Gly Val Lys Pro Gly

115 120 125

Leu Arg Pro Gln Ala Ile Ala Val Lys Leu Trp Asp Pro Glu Gly Ala

130 135 140

Gln Gly His Lys Glu Trp Leu Ala Glu Val Ile Phe Leu Gly Gln Leu

145 150 155 160

Arg His Pro Asn Leu Val Lys Leu Val Gly Tyr Cys Cys Glu Asp Glu

165 170 175

Asn Arg Leu Leu Val Tyr Glu Tyr Met Glu His Gly Ser Leu Glu Asn

180 185 190

His Leu Phe Lys Gln Ile Pro Ala Val Leu Pro Trp Ser Thr Arg Leu

195 200 205

Asn Ile Ala Val Gly Ala Ala Lys Gly Leu Ala Phe Leu His Asp Ala

210 215 220

Glu Lys Pro Val Ile Tyr Arg Asp Phe Lys Ala Ser Asn Ile Leu Leu

225 230 235 240

Asp Ser Asp Tyr Lys Ala Lys Leu Ser Asp Phe Gly Leu Ala Lys Asp

245 250 255

Gly Pro Glu Gly Asp Asp Thr His Val Ser Thr Arg Val Met Gly Thr

260 265 270

His Gly Tyr Ala Ala Pro Glu Tyr Ile Met Thr Gly His Leu Thr Ala

275 280 285

Lys Ser Asp Val Tyr Ser Phe Gly Val Val Leu Leu Glu Ile Leu Thr

290 295 300

Gly Arg Arg Ala Val Asp Lys Thr Arg Pro Asn Arg Glu Gln Ser Leu

305 310 315 320

Val Glu Tyr Ala Arg Pro Cys Leu Arg Asp Pro Leu Arg Leu Ile Arg

325 330 335

Ile Met Asp Pro Ala Leu Glu Gly Arg Tyr Ser Pro Ala Ala Ala Arg

340 345 350

Glu Ala Ala Ala Val Ala Tyr Arg Cys Leu Ser Gly Ser Pro Lys Asn

355 360 365

Arg Pro Asp Met Ser Ala Val Val Asp Ala Leu Glu Pro Leu Leu Val

370 375 380

Ala Thr Asp Asp Val Pro Leu Gly Pro Val Val Leu Phe Val Ala Pro

385 390 395 400

Asp Gln Glu Ala Asp Ala Ala Ala Ala Ala Asp Asp Asp Glu Asp Asp

405 410 415

Lys Ala Arg Arg Arg Gln Arg Arg Thr Arg Lys Asp Glu Gln His Arg

420 425 430

Arg Arg Ser Arg Leu Arg Thr Ser Pro Lys Gly Ser Pro Arg Lys Pro

435 440 445

Ala Val Ala Ala Ala Cys Arg Asn Glu Glu Phe Trp Val Trp His Val

450 455 460

Pro Ala Asp His Lys Ala

465 470

Claims

1. Use of OsRLCK239.1 or a homologue or a up-regulator thereof for:

improving the ability of plants to resist diseases and insect pests or preparing plants with improved ability to resist diseases and insect pests; or

Improving the plant type character of the plant or preparing the plant with improved plant type.

2. Use according to claim 1, wherein the pests comprise: bacteria; preferably, the bacteria comprise: bacterial blight (Xanthomonas oryzae pv. oryzae, Xoo), bacterial leaf spot (Xanthomonas oryzae pv. oryzae, oc), bacterial brown spot (Pseudomonaspora oryzae pv. syringae Van Holl) and bacterial rice basal rot (Erwinia chrysogenemia pv. zea victoria).

3. Use according to claim 1, wherein the pests comprise: an insect; preferably, the insects are hemipteran insects; more preferably insects of the family Nilaparvata, such as Nilaparvata lugens, Sogatella furcifera and Laileria laevigata; more preferably, OsRLCK239.1 or a homologue thereof improves the ability of a plant to resist a pest by reducing the feed intake of an insect.

4. The use according to claim 1, wherein the plant type trait of the modified plant or the production of a plant type modified plant comprises:

reducing the plant height of the plant or preparing the plant with reduced plant height;

promoting the leaf erection of plants or preparing plants with upright leaves; or

Increasing the aspect ratio of plant grain or making plants with increased grain aspect ratio.

5. The use according to claim 1, wherein OsRLCK239.1 is:

(a) 2, as shown in SEQ ID NO; or

(b) A protein which is formed by substituting, deleting or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO. 2, has the same function with the protein (a) and is derived from the protein (a); or

(c) Protein derived from (a) and having an amino acid sequence which is more than 80% identical to the amino acid sequence defined in (a) and has the same function as the protein of (a); or

(d) A protein fragment of SEQ ID NO 2 having the function of the protein of (a).

6. The use according to claim 1, wherein said plant comprises: a gramineous plant; preferably, the gramineae comprises: rice, sorghum, corn, barley, wheat, oats, rye.

7. The use of claim 1, wherein the up-regulator of OsRLCK239.1 comprises: an expression construct, expression cassette or expression vector of OsRLCK239.1; a molecule that interacts with OsRLCK239.1 to promote its expression or activity.

8. A method of increasing the ability of a plant to resist a pest or improving the plant type of a plant, or producing a plant with increased ability to resist a pest or improved plant type, the method comprising: increasing expression or activity of OsRLCK239.1 or a homologue thereof in a plant.

9. The method of claim 8, wherein the method comprises: transferring a polynucleotide or construct encoding OsRLCK239.1 or a homologue thereof into a plant; or

Administering an up-regulator of OsRLCK239.1 to a plant, thereby increasing expression or activity of OsRLCK239.1 or a homologue thereof in the plant.

10. The method of claim 9, wherein said method comprises the steps of:

(i) providing agrobacterium carrying an expression vector, wherein the expression vector comprises a polynucleotide encoding OsRLCK239.1 or a homolog thereof;

(ii) transferring said polynucleotide encoding OsRLCK239.1 or a homologue thereof into a plant using Agrobacterium.

11. The method of claim 8, wherein said plant comprises: a gramineous plant; preferably, the gramineae comprises: rice, wheat, barley, corn, sorghum.

12. The method of claim 8, wherein the pest comprises: an insect; preferably, the insects are hemipteran insects; more preferably insects of the family Nilaparvata, such as Nilaparvata lugens, Sogatella furcifera and Laileria laevigata; more preferably, OsRLCK239.1 or a homologue thereof improves the ability of a plant to resist a pest by reducing the feed intake of an insect.

13. The method of claim 8, wherein modifying the plant type trait of the plant or producing a plant type modified plant comprises:

14. Use of OsRLCK239.1 or homologues thereof or a polynucleotide encoding same as a molecular marker for identifying plant type traits or ability to resist pests in plants.

15. A plant cell expressing an exogenous osrlck239.1 or a homologue thereof, or comprising an exogenous expression cassette for osrlck239.1 or a homologue thereof; preferably, the expression cassette comprises: a promoter, a gene encoding OsRLCK239.1 or a homologue thereof, a terminator; preferably, the expression cassette is comprised in a construct or expression vector.